31P NMR and saturation transfer studies of the effect of Pb2+ on cultured osteoblastic bone cells.

T L Dowd,J F Rosen,R K Gupta

doi:10.1016/s0021-9258(17)45291-4

T L Dowd, J F Rosen + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)45291-4

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Abstract

The mechanism of lead toxicity at the cellular level remains unknown, although an effect of lead on intracellular Ca2+ has been described. Since bone is a major target for lead, we have investigated the effect of lead on bioenergetic rates and on the intracellular free Mg2+ concentration in cultured osteoblastic bone cells. Using 31P NMR and the saturation transfer technique we have detected a sizable (18%) transfer of saturation from gamma ATP to Pi in a perfused osteoblastic osteosarcoma bone cell line, Ros 17/2.8, and have found a large (greater than 82%) reduction in the Pi----ATP rate upon treatment with 10 microM Pb2+. The NMR-measured unidirectional rate was much greater than the net rate of ATP synthesis through glycolysis and oxidative phosphorylation. By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment. In addition, from the chemical shifts of intracellular ATP resonances, we found a significant reduction of 21% in the intracellular free Mg2+ concentration upon Pb2+ treatment. The observed lead-induced reduction in ATP synthesis/utilization and the decrease in intracellular free Mg2+ may contribute to the impairment of bone formation during lead intoxication.

Highlights

31P NMR and Saturation Transfer Studies of the Effect of Pb2+ on Cultured Osteoblastic Bone Cells*
By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenaselphosphoglycerate kinase.The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment
In this paper we report an application of 31P NMR and the saturation transfer technique to determine whether Pb*+ affects the unidirectional rate of ATP synthesis in the intact osteoblastic osteosarcoma bone cell line Ros 17/2.8

Summary

PROCEDURES

17/2.8, an osteoblastic osteosarcoma bone cell line, was maintained and grown to confluence in 75-cm* flasks with. Ham’s F-12 nutrient medium supplemented with 5% fetal bovine serum. This cell line is highly differentiated and expressive of the osteoblastic phenotype [22]. Measurements of the net rate of aerobic glycolysis were carried out in cells grown on 25.cm flasks at 37 “C. Cells in three flasks were incubated with 10 pM Pb” for 2 h prior to the measurements. Total protein was measured using the Biuret method. Oxygen consumption, on both control and Pb’+-treated cells, was measured on perfused, oxygenated samples using a Clark Electrode and a tissue oxygen consumption chamber (Yellow Springs Instruments Co.)

NMR spectra were collected on a Varian

Pi resonance is observed if the rate of exchange of intracellular

Quantitation of Intracellular

NMR spectra of untreated

DISCUSSION